An important role for RPRD1B in the heat shock response

During the heat shock response (HSR), heat shock factor (HSF1 in mammals) binds to target gene promoters, resulting in increased expression of heat shock proteins that help maintain protein homeostasis and ensure cell survival. Besides HSF1, only relatively few transcription factors with a specific role in assuring correctly regulated gene expression during the HSR have been described. Here we use proteomic and genomic (CRISPR) screening to identify a role for RPRD1B in the response to heat shock. Indeed, cells depleted for RPRD1B are heat shock sensitive and show decreased expression of key HSPs. These results add to our understanding of the connection between basic gene expression mechanisms and the HSR.

Vinculin is used as a loading control. (B) Immunofluorescence analysis of HSF1 nuclear foci formation. (C) qPCR quantification of nascent RNA near the 5'-end or the 3'-end of the HSPH1 gene, relative to GAPDH, normalized to the control. (D) qPCR quantification of RPRD1B mRNA, relative to GAPDH, normalized to the control. Average of three biological replicates; error bars indicate ±SD. The experiments were carried out in MRC5-VA cells. For the western blot and the immunofluorescence analysis, siRPRD1B was depleted either with a mix of four siRNAs (pool) or two single siRNAs (#1 and #2). For the qPCR, siRPRD1B was depleted with a mix of siRNAs (#1 and #2).    Vinculin is used as a loading control. HSF1 and RPRD1B blots were used also in Figure S6. (A, bottom) Quantification of fluorescence intensity of the pSer5 and pSer2 markers normalized to vinculin, average of two biological replicates.
37 o C for 11 to 13 days. Colonies were fixed by 4 % (v/v) formaldehyde and stained with a 0.1 % (w/v) crystal violet solution. Colonies from four biological replicates (each seeded into triplicate wells) were counted using a GelCount TM colony counter (Oxford Optronix Ltd) and normalised to untreated.
Proteins were separated on 4%-15% TGX gels (BioRad, 5671084) and transferred either to a nitrocellulose membrane (GE Healthcare Life Sciences, 10600002) or to a PVDF membrane (Merck Life Science, IPFL00010). Membranes were blocked respectively in 5% (w/v) skimmed milk in PBS supplemented with 0.1% (v/v) Tween20 (PBST) or in Intercept (PBS) blocking buffer (LI-COR, 927-70001) for 1 h at room temperature. Incubation with primary antibodies was carried out in PBST or Intercept (PBS) blocking buffer supplemented with 0.1% (v/v) Tween20 overnight at 4°C. Membranes were washed several times in PBST, incubated either with HRP-conjugated or LI-COR fluorescent dyeconjugated secondary antibodies diluted respectively in PBST or Intercept (PBS) blocking buffer supplemented with 0.1% (v/v) Tween20 for 45 min at room temperature and washed several times in PBST. Signal detection was obtained either using SuperSignal West Pico PLUS (Thermo Fsher Scientific, 34577) and Radiance plus (Azure Byosystems, AC2103) as ECL reagents, or Odyssey CLx

RT-qPCR
Total RNA was extracted using RNeasy mini kit (QIAGEN, 74106) following manufacturer instructions including an on-column DNase digestion (QIAGEN, 79254). 1 µg of RNA was used for Reverse transcription with TaqMan Reverse Transcription Reagents (Thermo Fisher Scientific, N8080234) according to manufacturer instructions. For detection of nascent RNA, random hexamers were used in the reverse transcription reaction and for the detection of mRNA, oligo dT primers. cDNA was amplified on a CFX384 Touch Real-Time PCR Detector (BioRad 1855485) using iTAq Universal SYBR Green Supermix (BioRad, 172-5124) with the following conditions: 5 min denaturation at 95°C and 39 cycles of 10 s denaturation at 95°C, 10 s annealing at 58°C, and 20 s extensions at 72°C. Primers amplifying GAPDH mRNA were used for normalization purposes. All primer sequences are listed in Supplementary Table S7 TTchem-seq The TTchem-seq was carried out as described in Gregersen et al., (2020) (Gregersen et al., 2020) in two biological replicates. MRC5-VA cells transfected either with a mix of two single RPRD1B siRNAs or control siRNA were seeded at density of 8 X10 6 /plate and were exposed to 2 hours of HS. The RNA was labelled in vivo with 1 mM 4SU (Glentham Life Sciences, GN6085) for 10 minutes prior to the addition of TRIzol (Thermo Fisher Scientific) that was used to stop the reaction at the desired time point. RNA was extracted according to manufacturer instructions.
As a control for equal sample preparation, we spiked-in 4-thiouracile (4TU) labelled RNA from S. cerevisiae (strain BY4741, MATa, his3D1, leu2D0, met15D0, ura3D0). The yeast culture was grown overnight in YPD medium, diluted to OD600 of 0.1 and grown to mid-log phase (OD600 of 0.8) and labelled 5 min with 5 mM 4TU (Sigma-Aldrich, 440736). Total RNA was extracted with PureLink RNA Mini kit (Thermo Fisher Scientific, 12183020) following the enzymatic protocol. 100 µg of human 4SU labelled RNA was spiked-in with 1 µg of 4TU-labelled yeast RNA and brought to a total volume of 100 µl with water. The mix was fragmented by adding 20 µl 1M NaOH and incubating in ice for 20 min. 80 µl of 1M Tris-HCl pH 6.8 were added to stop the fragmentation and samples were cleaned up twice with Micro Bio-Spin P30 Gel Columns (BioRad, 7326250) following manufacturer instructions. Biotinylation of 4SU and 4TU residues was carried out in a total volume of 250 µl 10 mM Tris-HCl pH 7.4 and 1 mM EDTA, containing MTSEA biotin-XXlinker (Biotium, BT90066) for 30 min at room temperature in the dark. The RNA was then purified by phenol:chloroform extraction, denatured 10 min at 65°C and added to 200 µl µMACS Streptavidine MicroBeads (Miltenyi Biotec, 130-074-101). After 15 min incubation at room temperature, the mix was loaded to a µColumn in the magnetic field of a µMACS magnetic separator. The beads were washed twice in a buffer containing 100 mM Tris-HCl pH7.4, 10 mM EDTA, 1M NaCl and 01% Tween20.

mRNA-seq
MRC5-VA cells transfected either with a mix of two single RPRD1B siRNAs or control siRNA were exposed to HS for 2h and then harvested by scraping in pre-warmed PBS and spun down. The pellets were then snap-frozen in liquid nitrogen and quickly defrosted for RNA extraction with RNeasy mini kit (QIAGEN, 79254) following manufacturer instructions including an on-column DNase digestion (QIAGEN, 79254). The RNA was used for library preparation.

Library preparation
For the TTchem-seq experiment 30 to 100 ng (ask ASF) of 4SU/4TU labelled RNA were used for library preparation with the KAPA RNA HyperPrep kit (Roche, 08098107702) following manufacturer instructions with modifications as previously described (Tufegdzic Vidakovic et al., 2020). Briefly, the fragmentation step was omitted and the RNA, resuspended in FPE Buffer, was denatured at 65°C for 5 min. The two SPRI bead purifications were carried out, respectively, with a bead-to-sample volume ratio of 0.95x and 1x. For the mRNAseq experiment, 200ng of purified RNA per sample were used to prepare polyA+ mRNA libraries with KAPA mRNA HyperPrep kit (Roche, 08098123702) following manufacturer instructions. The libraries were then sequenced with single end 75bp reads on the Hiseq2500, with 50,000,000 reads per sample.

Chromatin fractionation
The cells were harvested by scraping in PBS, washed once with PBS and spun down. The pellets were then snap-frozen in liquid nitrogen. The quickly defrosted pellets were used to obtain a soluble fraction

SILAC-based method for quantitative proteomic analysis
For mapping the RNAPII interactome in the presence of HS and DRB, MRC5-VA were cultured in SILAC light media or heavy media for 3 weeks. 97% efficiency of isotope incorporation was confirmed by mass spectrometry after 2 weeks in culture. The cells were then incubated with DRB either alone or in combination with HS before chromatin fractionation and the chromatin fraction was used for the immunoprecipitation (IP). 1 mg of chromatin fraction was used per IP (all samples were adjusted to the same volume, typically 800-950 µl). For immunoprecipitation of RPB1 with 4H8 antibody, 100 µl of packed Protein G agarose beads (Thermo Fisher Scientific, 20397) per sample were prepared by washing twice in PBS supplemented with 0.05% Tween and then coupling to 30 µl of 4H8 antibody (1 mg/ml stock) for 1 h on a turning wheel at room temperature. The beads were then washed twice in PBS plus 0.05% Tween20 and once in IP buffer (20 mM Hepes-KOH pH 7.5, 1.5 mM MgCl2, 10% glycerol, 150 mM NaCl, 0.05% NP-40) containing phosphatase and protease inhibitors. The beads were then resuspended in IP buffer containing phosphatase and protease inhibitors and added to the samples, to yield a 1 ml total reaction volume. The samples were incubated on a turning wheel in the cold room for 3 hours, and then centrifuged at 500 g at 4°C for 3 min. Supernatant (unbound fraction) was removed and saved, and the beads were washed 3 times in IP buffer. After the last wash, the beads were resuspended in 200 µl of IP buffer and loaded onto a Pierce TM Spin Columns-Screw cap (Thermo Fischer scientific, 69705) and spun 1 minute at 14000 rpm, the flowthrough was discarded. To elute the immunoprecipitated proteins, 50 µl of 2X Laemli buffer were added to the column, which were vortexed and boiled at 98°C for 5 min. The columns were centrifuged at 14000 rpm for 2 min and the elution was collected and used for western blot and mass spectrometry analysis. For mass spectrometry, light isotope-labeled samples and heavy isotope-labeled samples were mixed before loading on SDS-PAGE gel, for example, light labeled DRB-HS sample was mixed with heavy-labeled DRB sample; heavylabeled DRB sample was mixed with light-labeled DRB-HS sample. The mixed samples were run around 10 mm into the fixed 10% NuPAGE Bis-Tris and stained with Instant Blue (Expedeon, ISB1L).

Mass spectrometry
Each lane of the submitted Coomassie-stained SDS-PAGE gel was excised into ten equal bands along its entire length using a scalpel. Twenty bands were transferred to separate 1.5 ml tubes and de-stained with successive washes of 100 mM aqueous ammonium bicarbonate (AMBIC) solution followed by ethanol solution. Proteins were simultaneously reduced and alkylated by incubating bands in aqueous Thermomixer (70 ºC, 5 minutes, 1,000 rpm). Gel bands were washed by immersion in 50 % ethanol solution containing 50 mM AMBIC on a Thermomixer (22 ºC, 15 minutes, 1,000 rpm) then dehydrated by immersion in 100 % ethanol solution. Solutions were discarded after each incubation. A 20 µg vial of lyophilized trypsin (Pierce, MS Grade, 90057) was re-suspended in 50 mM acetic acid then diluted with 50 mM HEPES to produce a 2.5 ng/µl trypsin solution. 100 µl (250 ng) of trypsin was added to each gel band and the tubes were incubated on a Thermomixer (37 ºC, overnight, 1,000 rpm). The following morning, 50 µl of aqueous 25 % acetonitrile solution was added to each sample for peptide extraction. Tubes were placed in an ultrasonication bath for 5 minutes then the solution was transferred to new 1.5 ml tubes labelled "peptides". This extraction was repeated with a second 50 µl aliquot of aqueous 25 % acetonitrile solution and combined with the first. A final extraction using 50 µl of 100 % acetonitrile solution was performed. Peptide samples were dried via vacuum centrifugation then stored at -80 ºC.
Dried peptide samples were re-suspended in 200 µl of 0.1 % formic acid and loaded onto preequilibrated Evosep tips (Evosep, Denmark) using gentle centrifugation. An Evosep One robot loaded samples onto a C18 column (length 15 cm) and peptides were separated using the Evosep pre-defined 44-minute method. Eluted peptides were subsequently ionized and analyzed on a Q-Exactive orbitrap mass spectrometer (Thermo Scientific, USA) using a top 10 data-dependent acquisition method with settings: MS1 70k resolution, 1e6 AGC target, 250 millisecond maximum IT, 350-1800 m/z scan range, profile mode; MS2 35k resolution, 1e5 AGC target, 60 millisecond maximum IT, loop count 10, 200-2000 m/z scan range, NCE 33, profile mode. Forty.raw files were produced (one for each gel band).

Generation of Cas9-MRC5VA cell line
pCW57.1 dox inducible CAS9 vector and pLX-sgAAVS1 was purchased from Addgene. Cas9-MRC5VA cell lines were created by transduction of dox inducible flag-Cas9 vector. The cells were selected with Hygromycin for a week and plated at low density for clonal selection in a 15 cm dish.
Several clones were recovered and tested for the expression of flag-Cas9 by western blot analysis. The clone with the strongest expression of flag-Cas9 was selected and further used in the screen.

Optimization of Cleavage efficiency by Cas9
For optimizing the cleavage efficiency by Cas9, virus expressing sg RNA targeting the AAVS1 locus was transduced to Cas9-MRC5VA cell line. The virus was transduced at 20% transduction efficiency.
After 3 days of selection with blasticidin, Cas9 was induced with 1 µg/ml of doxycycline. Genomic DNA was extracted from cells after 1, 2-, 3-, 4-and 5-days post CAS9 induction. The AAVS1 locus was amplified using primers flanking the cleavage site (R-CCCCGTTCTCCTGTGGATTC, F-ATCCTCTCTGGCTCCATCGT). TIDE online software was used to detect the indels.

Virus production
pLX-sg-non targeting-A bunch of 30 non targeting plasmids was kindly provided by Paola Scaffidi. The Human CRISPR nuclear sub pool library was purchased from Addgene (Wang et al., 2014). Lentivirus of the nuclear sub pool was generated by co-transfection of the nuclear library sub pool with VSVG, GAG-POL and REV packaging plasmids into 293T cells using PIE (Polyethylenimine, Polysciences PIE-23966-1) transfection reagent. The media was refreshed once after 24 hours. 48 hours post transfection the viral supernatant was collected by passing it through 0.45 µm filters.

Pooled screening
The screen was performed in duplicates. 200 million target cells were transduced at 20% transduction efficiency with nuclear library viral pool in media containing 4µg/ml Polybrene (Sigma-Aldrich, H9268). After 3 days of selection with blasticidin, time zero (T0) cells were collected for genomic DNA extraction. Cas9 was induced with doxycycline (1µg/ml) for 5 days. CAS9 induced cells were split into two arms, untreated (UT) and heat shock (HS). The cells of the heat shock arm was incubated at 42 O C for 4 hours. Cells were passaged every three days and after 10 days cells were harvested for genomic DNA extraction. To prepare the sgRNA libraries for NGS, a two-step nested PCR-based approach described by Wang, T et al was followed. Genomic DNA was isolated from 40 million cells. To ensure efficient amplification of the sgRNAs, 60 first PCR reactions were run for each sample using Phusion DNA polymerase and a maximum of 1 µg gDNA in 50-µl reactions. Following the first PCR round, all reactions were pooled and 2 µl was used as template for the second PCR. Final products were pooled, run on a 2% agarose gel, excised, and purified using a QIAquick gel extraction kit (Qiagen, 28706) before sequencing. The libraries were analysed on a BioAnalyzer 2100 chip (Agilent) and then sequenced on an Illumina HiSeq 4000 platform using custom primers, generating ~30-50 million reads per sample.

Immunofluorescence staining
MRC5-VA cells were transfected with the indicated siRNAs according to siRNA interference protocol and then seeded on coverslips in a 6-well plate one day before harvesting. The cells were fixed with 3% paraformaldehyde in PBS for 10 minutes at room temperature. This was followed by permeabilization for 5 minutes with PBS supplemented with 0.5% Triton X-100. Cells were washed with 1 mL PBS for twice and blocked with PBS containing 5% milk for 30 minutes at room temperature. Primary antibodies (HSF1 Enzo Life Sciences Cat # ADI-SPA-901, 1:150) were diluted in PBS and incubated 30 minutes at room temperature. Cells were then washed twice in PBS supplemented with 0.1% Triton X-100 and incubated with secondary antibody in PBS (Donkey anti-Rabbit Alexa Fluor 488, Cat # A-21206 Thermo Fisher Scientific) for 30 minutes at room temperature. Cells were washed twice with 1 mL PBS supplemented with 0.1% Triton X-100 and the coverslips let air dry before mounting on glass slides with antifade mounting medium with DAPI (Vector Laboratories, H-1700). Images were acquired using micro-manager software (Edelstein et al., 2014) on a Zeiss Observer.Z1 wide-field microscope, equipped with a Plan-NEOFLUAR 20X/0.5 dry and a Plan-APOCHROMAT 40X/1.3 oil immersion objectives, selective bandpass filters for DAPI, GFP, RFP, Cy5, and a Hamamatsu Orca Spark CMOS camera. Images were visualized using FIJI open-source software (Schindelin et al., 2012).

TT-seq and mRNA-seq read alignment and quantification
Reads were processed using the publicly available nf-core rnseq pipeline v3.3 with the STAR/RSEM option against human genome assembly GRCh38 and Ensembl release 104 transcript annotations.
HS conditions. Data are presented for i) all genes and ii) 280 genes shown to be induced between HS/non-HS control conditions in the mRNA-seq analysis (FDR<0.05, log2FC>1 and baseMean>100).

Splicing analysis
Identification and quantification of splice-variants was conducted using the software Whippet v0.11.1 (Sterne-Weiler et al., 2018) running on Julia v0.6.4. A splice index was created using Gencode v39 basic gene annotation and the GRCh38 genome sequence using Whippet's whippet-index.jl function with default settings except for "--suppress-low-tsl". Each sample fastq file was quantified for PSI (percent spliced-in) values in turn using the whippet-quant.jl function with default settings except for "--biascorrect". Differential splicing events were assessed between replicate groups in a pairwise fashion by comparing PSI values using the "whippet-delta" function ("--min-reads 5, --min-samples 3, -g 123456"). Thresholding of the differential splicing results was conducted using the Bioconductor package GeneStructureTools (Signal, 2021), filtering the data based on a minimum probability >=0.99, a change in PSI of >=0.3, a mean of at least 100 counts in one of the conditions to obtain a high confidence set.

sgRNA analysis
Raw reads from CRISPR libraries were trimmed to 20bp using cutadapt with the "--cut -<trim_size>" parameter. These were then mapped to the appropriate guide sequences using BWA (version 0.5.9-r16) (Li and Durbin, 2009) with the parameters "-l 20 -k 2 -n 2". sgRNA counts were obtained after filtering the mapped reads for those that had zero mismatches, and mapped to the reverse strand. The MAGeCK 'test' command (version 0.5.3) (Li et al., 2014) was used to perform the sgRNA ranking analysis between the relevant conditions with parameters "--norm-method total --remove-zero control".

CRISPR screen hit identification
sgRNAs depleted after gene KO were identified by comparing the normalized sgRNA counts in the Untreated (UT) population after CAS9 induction and Time zero (T0). Comparison of the UT with HS identified those sgRNAs which are depleted specifically in response to Heat shock treatment. Only sgRNAs with at least 10 raw counts in all the replicates at T0 were used for analysis. Raw reads for each sgRNA were normalized to total read counts for each sample, and the fold change between UT and HS was calculated for each replicate. Depleted sgRNAs showing a log2(FC) ≤ −1 in all the three triplicates were selected.
Variable modifications of protein N-terminal acetylation and methionine oxidation, and fixed modification of cysteine carbamidomethylation were set. A SwissProt Homo sapiens protein database (downloaded January 2019) was queried using the default 1 % FDR at both protein and peptide levels. Results .txt files from MaxQuant were further analyzed in Perseus software version 1.4.0.2 (Ref 2). Results were filtered to remove potential protein contaminants (including keratins), reversed sequences and "only-identified by site" hits. Protein intensities were log10 transformed and SILAC H/L ratios were log2 transformed.